The present invention relates to a method of producing IgG for medical applications. The method of the invention provides an IgG product having low anticomplementary activity (ACA).
IgG prepared from human plasma is widely used in the treatment of agammaglobulinaemia, idiopathic thrombocytopenic purpura and in the prophylaxis of certain diseases. IgG preparations are administered intramuscularly as well as intravenously.
Within the field of the art it is well known that isolated IgG preparations have marked anticomplementary activities (ACA). It has been shown that the components responsible for these activities are aggregates of IgG that form either spontaneously or as a result of the isolation procedure. These anticomplementary aggregates have been shown to be harmful in several clinical applications of the IgG products. For example, intravenous administration of IgG preparations can give rise to adverse side reactions, including anaphylactic shock.
Several solutions have been proposed to overcome the problem with ACA in IgG preparations. For example, in U.S. Pat. No. 3,966,906 a process is described for treating a crude gamma globulin fraction of serum with pepsin to disaggregate IgG and reduce anticomplement activity. However, the therapeutic effect provided by such a preparation is unacceptably short since it is rapidly excreted. Another drawback with the pepsin treated immunoglobulins is that their Fc binding capacity is lower than for native immunoglobulins.
Attempts have been made to stabilise pepsin treated IgG preparations, such as by polyethylene glycol (PEG) see for example WO 86/06993.
To solve the problem with high ACA activity it has been proposed to chemically modify the IgG preparations. For example, in U.S. Pat. No. 3,902,262 a portion of the disulphide linkages of the IgG molecule is reduced to xe2x80x94SH groups and then the xe2x80x94SH groups are alkylated.
For obvious reasons, it would be desirable to have an IgG product which is free from enzymatic and other chemical modification and to be as close to native as possible. A method fulfilling these criteria has been described in xe2x80x9cAn improved chromatographic method for production of IgG from human plasmaxe2x80x9d by I. Andersson, L-O Lindquist, J. Berglxc3x6f, presented at the xe2x80x9cXXIII Congress of the ISBTxe2x80x9d, Amsterdam, The Netherlands, Jul. 2-8, 1994). However, this procedure also shows unsatisfactory high ACA-levels and does not fulfil the FDA and EU requirements for intravenous drugs.
Known methods for the manufacture of IgG compositions typically comprise several steps selected amongst:
a) buffering plasma, for instance either by subjecting the plasma to an appropriate gel filtration chromatography or by diafiltration;
b) removing euglobulins, such as by precipitation;
c) removing the albumin fraction (albumin), for instance by binding albumin and the like to an anion exchanger leaving the IgG in the unbound fraction (IgG fraction);
d) purifying, after removal of euglobulins and albumin, the IgG fraction obtained in step (c) on an anion exchanger and collecting the unbound fraction;
e) purifying the plasma fraction obtained in step (d) on a cation exchanger and collecting the bound fraction (adsorption and release of IgG);
f) concentrating the IgG enriched plasma fraction obtained in step (e) (IgG released from the cation exchanger), preferably by ultrafiltration;
g) inactivating viruses by adding virus inactivation chemicals, preferably a solvent/detergent (S/D) solution, to an IgG enriched plasma fraction, for instance the fraction obtained in step (f);
h) removing the virus antiviral chemicals added in step (g), preferably by adsorbing IgG to a cation exchanger and releasing and collecting the bound fraction;
i) concentrating the bound fraction collected in step (h), for instance by ultrafiltration;
j) formulating of the fraction concentrated in step (i);
k) sterile filtration of the formulated IgG obtained in step (k).
The present invention provides a solution for producing IgG products having reduced ACA, by modifying earlier known methods.
Thus in a first aspect the invention relates to a method of producing IgG from plasma for medical applications, comprising at least: (ixe2x80x2) removal of albumin resulting in an IgG fraction, (iixe2x80x2) purifying IgG from an IgG fraction, which is derived from the IgG fraction obtained in step (ixe2x80x2), by adsorbing IgG to a cation exchanger and collecting the adsorbed IgG fraction, and (iiixe2x80x2) virus inactivation in an IgG fraction derived from the IgG fraction collected in step (iixe2x80x2). The method is characterized in
(I) concentrating the IgG fraction obtained in step (ixe2x80x2),
(II) adjusting pH to 4xc2x10.1 of the IgG fraction released from the cation exchanger used in step (iixe2x80x2), and preferably maintaining the pH below 6.0 during the remaining steps of the method; and
(III) carrying out the virus inactivation (step iiixe2x80x2) by using virus inactivation chemicals, preferably a solvent/detergent (S/D) solution, at a temperature of 30xc2x0 C.xc2x12xc2x0 C. for at least 4 hours.
Adjustment of pH to around 4 in (II) permits virus inactivation to be carried out at around 30xc2x0 C. In the corresponding earlier process, in which pH was 5,5, the level of the proteolytic activity at 30xc2x0 C. was unacceptable.
A preferred variant of the process according to the invention comprises steps (a)-(k) above in which the albumin removal step (c) corresponds to (ixe2x80x2), the cation exchange step (e) corresponds to (iixe2x80x2), and the virus inactivation (g) corresponds to (iiixe2x80x2).
The purification and/or removal steps above are preferably run as chromatography. Appropriate separation media used in these steps are hydrophilic in the sense that they are able to expose surfaces carrying hydrophilic groups, such as hydroxy, amido etc., to the liquid sample containing IgG. Appropriate separation media may be found amongst those that are based on synthetic polymers and/or biopolymers (for instance polysaccharides) carrying hydrophilic groups, as referred to above. Depending on where in the process the media is to be applied they may be uncharged, or may carry positively charged (e.g. ammonium groups) and/or negatively charged groups (e.g. carboxy groups and sulphonic acid groups). The following chromatographic media are preferred:
step a): Sephadex G25;
step c): DEAE Sepharose FF;
step d): Q Sepharose FF;
step e): CM Sepharose FF;
step h): CM Sepharose FF.
Sephadex and Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden) are based on cross-linked dextrand and agarose, respectively. DEAE means that the base matrix (cross-linked dextran is substituted with diethylaminoethyl groups. Analogously Q stands for quaternary ammonium groups, and CM for carboxy methyl groups.
The concentrating according to (I) is preferably performed immediately after albumin removal (for instance after step c as defined above) by ultrafiltration, to less than or equal to the volume of the starting plasma.
To be able to use an acetate buffer in the step for removal of virus inactivation chemicals (step h), the ionic strength is adjusted to about 1.40 mS before this step.
In a preferred embodiment, the method also comprises, in step (i) lowering of ionic strength to 0.5 mSxc2x10.1, preferably by diafiltration against distilled water.